Functional analysis of the interleukin-27-induced antigen processing and presentation on oral and intestinal mucosal surfaces

在人类免疫系统中，(外来)抗原与MHC I类和II类受体结合，并被呈递给免疫细胞。虽然MHCI受体在几乎所有的体细胞上都有表达，但MHCII受体主要存在于专业抗原提呈细胞(APC)的表面。除了免疫系统的特殊细胞外，粘膜表面的细胞，如消化道的肠上皮细胞(IEC)和口腔的口腔上皮细胞(OEC)在免疫防御中也发挥着重要作用。它们不断地与共生细菌和致病细菌接触，从而以平衡的方式产生促炎和抗炎细胞因子。如果这种微调反应受到干扰，可能会导致过度的免疫反应，进而导致慢性炎症，如炎症性肠病(IBD)和慢性牙周炎(CP)。白介素27(IL-27)是一种在多种T细胞分化过程中起重要作用的细胞因子，其在IBD患者中的表达增加。自身分析表明，IL-27对肠屏障也有保护作用，并诱导IEC表达抗炎和抗菌蛋白。初步的微阵列分析表明，IL-27上调了MHC II类表达所必需的主要转录因子CIITA以及IEC和OEC中MHC基因座的其他基因，从而使这些细胞有可能成为非专业的APC。在这些初步研究的基础上，本方案的目标是对IL-27诱导的IEC和OEC的抗原提呈进行详细的功能分析。进一步鉴定直接或间接参与抗原处理和提呈的基因(如免疫蛋白酶体)，并对所涉及的信号转导途径和调节DNA元件进行详细分析。为此，将应用蛋白质印迹、siRNA转染、EMSA、CHIP、Co-IP、免疫荧光和荧光素酶检测等分子生物学技术。为了分析IL-27在诱导MHC分子和肠道抗原提呈中的作用，将建立上皮特异性IL-27受体基因敲除小鼠(使用Cre-lox系统，在上皮特异性绒毛蛋白启动子控制下表达Cre重组酶)，并将在结肠炎T细胞过继转移模型中进行分析。此外，来自健康受试者、IBD和CP患者的组织活检将通过免疫组织化学分析MHC分子的表达。使用针对细胞类型特定标记的抗体的双重染色将被用于确定存在MHC表达的细胞亚型。这一建议的结果将有助于我们了解IBD和CP的发病机制，并有助于更好地理解粘膜表面细胞作为非专业性APC的作用。

In the human immune system, (foreign) antigens are bound by MHC class I and class II receptors and are presented to immune cells. While MHCI receptors are expressed on nearly all somatic cells, MHCII receptors are mainly found on the surface of professional antigen-presenting cells (APCs). Besides specialized cells of the immune system, cells of mucosal surfaces such as intestinal epithelial cells (IEC) of the digestive tract as well as oral epithelial cells (OEC) of the oral cavity play an important role in immune defence. They are constantly in contact with commensal and pathogenic bacteria and produce thereupon pro- and anti-inflammatory cytokines in a balanced manner. If this fine-tuned reaction is disturbed, this may lead to excessive immune responses and subsequently to chronic inflammation such as inflammatory bowel disease (IBD) and chronic periodontitis (CP). Interleukin-27 (IL-27) is a cytokine that plays an important role in the differentiation of several T cell types and its expression is increased in IBD patients. Own analyses have shown that IL-27 also is protective for the intestinal barrier and induces expression of anti-inflammatory and anti-bacterial proteins in IEC. Preliminary microarray analyses indicated that IL-27 up-regulates CIITA, the master transcription factor necessary for MHC class II expression, as well as other genes of the MHC locus in IEC and OEC thereby potentially enabling these cells to act as non-professional APCs. Based on these preliminary studies, the goal of this proposal is a detailed functional analysis of the IL-27-induced antigen presentation in IEC and OEC. Further genes directly or indirectly involved in antigen processing and presentation (e.g. immunoproteasome) are to be identified and the involved signal transduction pathways and regulatory DNA elements will be analyzed in detail. For this purpose, molecular biology techniques such as western blot, siRNA transfection, EMSA, ChIP, Co-IP, immunofluorescence and luciferase assays will be applied. To analyze the role of IL-27 in inducing MHC molecules and intestinal antigen presentation, epithelial-specific IL-27 receptor knockout mice will be generated (using the Cre-lox system with Cre recombinase expressed under control of the epithelial-specific villin promoter) and will be analyzed in the T cell adoptive transfer model of colitis. Moreover, tissue biopsies from healthy subjects and IBD and CP patients will be analyzed by immunohistochemistry for expression of MHC molecules. Double stainings with antibodies against cell type specific markers will be used to determine the cellular subtype in which MHC expression is present. The results of this proposal will contribute to our knowlegde of IBD and CP pathogenesis and will help to better understand the role of cells on mucosal surfaces as non-professional APCs.